Nicotinic acetylcholine receptors (nAChR) are widely expressed as multiple subtypes. Different subtypes have critically important, and distinct, roles in normal and disease physiology. nAChR research has long been stymied by a lack of sufficiently subtype-selective compounds. Cone snails are specialized marine predators that hunt a wide variety of prey. They have evolved a rich variety of peptides (conotoxins) that selectively target a wide range of receptors and ion channels, and therefore provide a uniquely extensive source of lead compounds. Those that target nAChR are referred to as ?-conotoxins (?-CTxs). Previous awards have been used to discover, develop, and deploy several ?-CTxs that are now standard tools, widely used to study nAChR subtype roles in health and disease. Despite this, an estimated 95% of native ?-Ctxs remain to be discovered, and most nAChR subtypes lack sufficiently selective ligands. This renewal proposal will accelerate exploitation of the ?-CTx resource, and develop antagonists with novel selectivities for physiologically- important nAChR subtypes. Aim 1 will develop antagonists for ?6?4 nAChR, which are present in dorsal root ganglia neurons and are thought to be mechanistically important in pain responses. Aim 2 will focus on ?7?2 nAChR, a subtype implicated in neuropsychiatric disorders. Aim 3 will develop ligands targeted to a non- canonical subunit interface, producing ?3?4?5 nAChR selective antagonists. This subtype is an important contributor to aspects of nicotine addiction. These ligands will be utilized in studies funded by the current award and will be provided to others, enabling molecular dissection of nAChR subtypes in electrophysiological, behavioral and biochemical studies. Preliminary studies using an existing ?-CTx library prove the feasibility of the proposed approach. Multiple novel approaches have been adopted in this renewal application to increase the pace and precision with which novel ?-CTxs may be discovered and optimized. For example, optimized ?- CTx analogs will be generated through positional scanning mutagenesis, in which active sites are modified in accord with previously determined structure-function information from related toxins. The available ?-CTx library will be further expanded by transcriptome analysis, a technique that has recently been successfully applied to cone snail species. Peptides will be produced by solid-phase chemical synthesis and initially characterized on heterologously expressed receptors. These will include nAChR composed of concatenated subunits that provide precisely defined subunit stoichiometry and subunit order, and improved-throughput cell- line-based screening approaches. Newly developed peptides will be used to characterize native nAChR using high-content calcium imaging in DRG neurons. Continued funding will allow expansion of this award's current high impact (see Progress Report) by 1) developing ligands for multiple translationally-relevant nAChR subtypes, 2) enabling faster and more effective exploitation of the under-explored ?-CTx resource (now and in the future), and 3) continued supply of existing and novel ?-CTxs to the broader scientific community.